Methylation of arginine residues is a common post-translational modification whose functions are not well understood. Interestingly, a very large percentage of the arginine-methylated proteins identified to date are RNA binding proteins. The incidence of arginine methylated RNA binding proteins and the heightened importance of posttranscriptional gene regulation in trypanosomes suggest that arginine methylation is likely to be especially important in regulating gene expression in these organisms. In support of this hypothesis, disrupting expression of the TbPRMT1 protein arginine methyltransferase (PRMT) leads to a severe growth defect in procyclic form Trypanosoma brucei. Thus, in contrast to yeast, which exhibit no discernable phenotype upon genetic disruption of the major PRMT, T. brucei is an excellent model system for the proposed studies. In addition to the TbPRMT1 disruption studies, we have acquired novel preliminary data indicating that arginine methylation is widespread in T. brucei. We identified two biochemically distinct classes of PRMT activities and multiple proteins that act as substrates for these enzymes in vitro. We also determined that the mitochondrial RNA binding protein, RBP16, undergoes multiple, mutually exclusive methylation events. To begin to elucidate the cellular and biochemical functions of arginine methylation in trypanosomes, experiments proposed in Aim 1 will analyze the impact of arginine methylation on the prototypical methylprotein, RBP16. Trypanosomes harboring tagged RBP16 molecules mutated at relevant arginine residues will be analyzed in vivo to discern the effects of inhibiting methylation of specific arginine residues on mitochondrial import, regulation of mitochondrial RNA editing and stability, and protein-RNA and protein-protein interactions. In Aim 2, we will identify proteins that physically and/or functionally interact with TbPRMT1 using methylation assays and protein interaction screens. In Aim 3, we will analyze trypanosomes in which expression of TbPRMT1 or a putative Type II PRMT has been disrupted using RNA interference. The impact of PRMT disruption on 1) nucleocytoplasmic mRNA transport, 2) trans-splicing, and 3) cytoplasmic mRNA turnover will be determined. The proposed studies will provide important insights into genetic and cellular regulation in trypanosomes, and will shed light on novel functions of a common but poorly understood posttranslational modification in higher eukaryotes as well.